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The specificity profile for Cre reveals its relative preference for the canonical base at each position in the loxP site. We sought to develop a system for profiling recombinase specificity by identification of bona fide recombinase substrates from an enormous in vitro library of attainable targets. Recombinase specificity profiling of wild-type Cre. As well as, Rec-seq identified a contribution from a secondary residue previously unknown to participate in specifying positions 5-7. Ala substitution at Gln94 resulted in decrease specificity at positions 6 and 7 but compensatory will increase elsewhere (Fig. 2e), despite the fact that Gln94 does not directly contact the DNA, but as a substitute engages in hydrogen bonds with Gln9035. Together, Rec-seq profiling clarifies the various interactions that collectively outline Cre recognition at positions 5-7, and highlights the important roles of secondary and oblique interactions. These lengthy-vary effects can't be inferred from the Cre:loxP construction, highlighting the utility of unbiased, high-resolution specificity profiling. Determinants of Cre:loxP specificity recognized by Rec-seq on wild-type Cre and Ala-substituted Cre variants

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